Epimerdinoside a oral pharmaceutics containing the same and preparatory and determination methods

ABSTRACT

Epimeredinoside A extracted from  Epimeredi indica  root, oral pharmaceutics made up with the  Epimeredi indica  root extract and at least one pharmaceutical adjuvant, and preparatory method for the oral pharmaceutics. The root extract is the extracta sicca prepared by water extraction and concentration of  Epimeredi indica  root, and contains 0.10% to 1.50% of epimeredinoside A. The inventive pharmaceutics of  Epimeredi indica  root extract do not contain any hormone and progesterone is not needed to be taken to prevent a side effect after using the drug. The pharmaceutics has doubtless effect in treating female menopause.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of TCM pharmaceutics, mainly dealing with anti-female menopausal-syndrome-effective epimeredinoside A, and epimeredinoside A-contained pharmaceutics of Epimeredi indica extract and the pharmaceutics' preparatory method.

2. Background of the Related Art

Estrogen and its pharmaceutics have been applied widely for the treatment of menopausal syndrome for a long time. However, it is hard to gain acceptance by women due to its many side effects and adverse reaction, even leading to cancer. Therefore, there is no satisfactory clinical drug at present.

Epimeredi indica (L.) Rothmalex, Guang-Fang-Feng, also named Fang-Feng-Cao, is recorded in The Dictionary of Traditional Medicine and is the whole plant of Epimeredi indica in the Labiatae family. It has been used in the treatment of many disorders such as cold with fever, disgorging, abdominal pain, bones and muscles pain, pyocutaneous disease, eczema, hemorrhoids and so on. It is used in the formula of Guanfang Ganmao Pills recorded in Volume 20 of Zhong-Yao-Cheng-Fang-Zhi-Ji (the TCM Pharmaceutics of Patent Formula) published by the Ministry of Public Health of the People's Republic of China.

A new use for Epimeredi indica root has been announced in Chinese Patent No. 02110522.7 by the inventor. Epimeredi indica root has the effects of ameliorating ovary function and regulating estrogen and progestogen, therefore it can be used to prepare drugs and health care products to treat and prevent many diseases due to the imbalance of estrogen and progestrogen.

SUMMARY OF THE INVENTION

The present invention further develops pharmaceutics of Epimeredi indica root extract on the basis of Chinese Patent No. 02110522.7, about a novel oral pharmaceutics with clear active constituent and its content and stable quality.

The present invention announces all kinds of pharmaceutics related to any oral pharmaceutics composed of Epimeredi indica root extract and pharmaceutical adjuvant. This extract is obtained from extracts of Epimeredi indica root, after being extracted by water and concentrated by distillation, containing 0.10% to 1.50% of epimeredinoside A.

Pharmaceutical adjuvants involved in the present invention are all common adjuvants in regular pharmaceutics. The oral pharmaceutics are any oral dosage forms widely used in the medical area including hard capsule, soft capsule, granule, tablet, oral liquid and so on.

Another technical point announced in the present invention is the preparatory method of the extract and determination method of active constituents in it.

A preparatory method for Epimeredi indica root extracts of the present invention comprises the following steps:

1. The roots of Epimeredi indica were powdered. Then, a 10 times amount of water was added, and extraction conducted two times, for 1˜2 hours per time. After filtration, it was concentrated as extracta sicca to a density of 1.01 to 1.08 (25˜30° C.), then dried by spray or vacuum. The content of epimeredinoside A in this extract was 0.10 to 1.50% as determined by HPLC.

2. Proportions of extracts and adjuvants were mixed well to prepare various pharmaceutics conventionally by wet or dry granulation.

The content determination method of Epimeredinoside A in extracts of Epimeredi indica root of the present invention comprises the following steps of:

1. Apparatus and Materials:

-   -   Instrument: Agilent 1100 HPLC system     -   Standard: epimeredinoside A     -   Chemical reagents: methanol, acetonitrile, distilled water and         other reagents were HPLC grade     -   Sample: Extracts of Epimeredi indica root (Shanghai Yaogang         Biotechnology Ltd. Co.)

2. Chromatographic Conditions:

-   -   Chromatographic column: Discovery C₁₈ (250 mm×4.6 mm, 5 μm)     -   Mobile phase: acetonitrile:water=27:73     -   Flow rate: 1.0 ml/min     -   Column temperature: room temperature     -   Detection wavelength: 320 nm     -   Injection volume: 20 ul

3. Calibration Curve:

-   -   Preparation of standard stock solutions: The standard was         prepared by weighing 4.95 mg, and dissolving and diluting with         methanol in a 25 ml volumetric flask to obtain standard stock         solutions for the calibration curves.     -   The Calibration Curves: From the stock solution, 0.4, 0.8, 1.2,         1.6, and 2.0 ml were weighed, respectively, dissolved, and         diluted with methanol in 2 ml volumetric flasks to obtain         standard solutions at concentrations of 39.6 μg/ml, 79.2 μg/ml,         118.8 μg/ml, 158.4 μg/ml, and 198 μg/ml, respectively.

A total of 20 μL of each standard solution was subjected to HPLC quantitative analysis. A calibration curve was generated to confirm the linear relationship between the peak area ratio (Y axis) and the concentrations of the standard (X axis) in the test samples. The calibration curves were found to be linear and could be described by the regression equations Y=20.139X−154.35, with coefficient R²=0.9994. The range of calibration curves was 0.792-3.96 μg, and the retention time of epimeredinoside A was 9.55 min.

4. Sample Determination

Preparation of the standard solutions: The standard was accurately weighed, and dissolved and diluted with methanol in a volumetric flask to obtain standard solutions. A total of 20 μL of standard solution was subject to HPLC quantitative analysis and the peak area was recorded. The content of epimeredinoside A was calculated using the calibration curves accordingly, see FIG. 2.

Preparation of the sample solutions: The extracts of Epimeredi indica root (176.66 mg) were accurately weighted, and extracted by ultrasonication at room temperature for 2 times, then centrifuged. The supernatants were combined and diluted with water in a 10 ml volumetric flask. The solution was filtered through a syringe filter (0.45 μm).

The sample solutions were subjected to HPLC analysis as described above. The content of epimeredinoside A in the samples was calculated according to the calibration curves.

Formula for calculation is as follows: Y=20.139X−154.35

-   -   Y: value of peak area     -   X: value of sample concentration (μg/ml)

The content of epimeredinoside A in a sample is demonstrated as X*10/*amount of sample*100%.

The Epimeredinoside A used in the present invention is an active compound obtained from extracts of Epimeredi indica root through isolation and purification. Extracts of Epimeredi indica root were extracted with n-butanol. The soluble extracts were then chromatographed on macroporous resin and a C-18 silicon column, eluted with ethanol gradient, collected and assayed by TLC. The ethanol elute was concentrated to obtain epimeredinoside A. FIG. 2 is its chromatogram of HPLC. Its structure is showed as follows:

Validation of the HPLC methods for determination epimeredinoside A in the present invention:

(1) Calibration Curve:

-   -   Preparation of standard stock solutions: The standard was         prepared by weighing 4.95 mg, and dissolving and diluting with         methanol in a 25 ml volumetric flask to obtain standard stock         solutions for the calibration curves.     -   The Calibration Curves: From the stock solutions, 0.4, 0.8, 1.2,         1.6, and 2.0 ml were weighed, respectively, dissolved, and         diluted with methanol in 2 ml volumetric flasks to obtain         standard solutions at concentrations of 39.6 μg/ml, 79.2 μg/ml,         118.8 μg/ml, 158.4 μg/ml, and 198 μg/ml, respectively.

A total of 20 μL of each standard solution was subjected to HPLC quantitative analysis. A calibration curve was generated to confirm the linear relationship between the peak area ratio (Y axis) and the concentrations of the standard (X axis) in the test samples. The calibration curves were found to be linear and could be described by the regression equations Y=20.139X−154.35, with coefficient R²=0.9994. The range of calibration curves was 0.792-3.96 μg, and the retention time of epimeredinoside A was 9.55 min.

Peak area Number 1 2 3 4 5 Sample 39.6 79.2 118.8 154.4 198 concentration (μg/ml) Peak area (mAU) 612.811 1472.17 2234.391 3036.277 3802.776

Calibration of epimeredinoside A is given in FIG. 1.

(2) Precision

To imbibe a standard solution at a concentration of 0.198 mg/ml for precision study under the above HPLC chromatographic conditions, then inject the above standard solution six times consecutively.

Number Peak area X RSD (%) 1 3802.776 3815.223 0.824 2 3806.568 3 3879.024 4 3796.254 5 3802.456 6 3804.259

The results showed that the precision of this method is preferable.

(3) Stability

Peak area of standard solution was assayed at 0, 4, 8, 12 h with an injection volume of 20 ul per time.

Number 1 2 3 4 Peak area 3785.21 3749.56 3802.54 3855.23 Mean 3798.135 RSD (%) 1.16

(4) Reproducibility

Five samples that have the same batch number were prepared for measurement according to the criteria of the sample assay procedure mentioned above.

Peak area of epimeredinoside A in a sample solution was assayed with an injection volume of 20 μl.

Number 1 2 3 4 5 Peak area 522.824 531.245 536.258 522.356 514.252 Mean 525.387 RSD (%) 1.63

(5) Recovery

The determined samples were weighed accurately and the standard epimeredinoside A solutions were added into the samples accordingly, and the content of epimeredinoside A in samples were determined under the same conditions as described above.

Added/ Analysis/ NO. Sample/μg μg μg Recovery Average RSD(%) 1 38.643 31.68 68.495 97.400 98.292 5.26 2 38.643 31.68 66.455 94.500 3 38.643 39.6 72.922 93.199 4 38.643 39.6 74.8 95.600 5 38.643 47.52 99.362 102.552 6 38.643 47.52 91.764 106.500

The results showed that a sensitive and stable analysis method for the determination of Epimeredi indica Root Extract was established.

The inventive pharmaceutics of Epimeredi indica Root Extract do not contain any hormone. No progesterone is needed to be taken to prevent a side effect after using the drug. It is compatible for a female in menopause that the drug has doubtless effect in clinic, stability, controllable and safety. Furthermore, a new approach was provided for patients which need to use estrogen but with contraindication of hormone.

BRIEF DESCRIPTION OF THE VARIOUS VIEWS OF THE DRAWING

FIG. 1: Calibration curve of the epimeredinoside A;

FIG. 2: HPLC chromatogram of epimeredinoside A; and

FIG. 3: HPLC chromatogram of Epimeredi indica Root Extract.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Preparation of Epimeredinoside A

(1) The dried and powdered root of Epimeredi indica was extracted with 10 fold water for 2 hours, and filtered. The residue was extracted with 8 fold water for 2 hours again, and filtered. The filtrates were combined and evaporated under vacuum to afford Epimeredi indica Root Extracts.

(2) The 6 kg of Epimeredi indica Root Extracts was extracted with 10 fold water for 3 times, and the solvent was evaporated to 600 ml. The residues were extracted with aqua-saturated n-butanol for 3 times (400 ml/time). The n-butanol solvent was evaporated under vacuum. The extracts of n-butanol were dissolved in water and chromatographed in a macroporous resin column (AB-8, Nankai Chemistry Factory, Tianjin). The chromatographic column was eluted with gradient mixtures of 20%, 50% and 95% aqueous ethanol successively. The elutes of 50% ethanol were concentrated and then dissolved with 50% aqueous methanol. The samples of 50% methanol were chromatographed on a RP-C18 silica column, eluted with 50% aqueous methanol to produce epimeredinoside A.

The structure of epimeredinoside A was elucidated by UV, IR, ESI, HRESI, NMR, 2D-NMR (COSY, HMQC, HMBC, NOESY) data. Epimeredinoside A, mp 139˜142□, molecular formula of C₁₃H₄₀O₁₅ and the molecular weight 652, was isolated. The ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectral data of Epimeredinoside A (CDCl₃) was shown in Table 1.

TABLE 1 ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectral data of Epimeredinoside A (CDCl₃) Ferulic acid δC ΔH Aglycone ΔC δH 1 127.68 1 132.69 2 111.66 7.15(d, 2) 2 117.00 6.69(d, 2) 3 150.64 3 147.47 4 149.36 4 147.33 5 116.47 6.80(d, 8) 5 112.81 6.65(d, 8) 6 124.27 7.02(dd, 8, 2) 6 121.11 6.61(dd, 8, 2) 7 147.10 7.62(d, 16) α 36.71 2.80(t, 7) 8 115.28 6.39(d, 16) β 72.31 3.5-4.2 9 169.07 OCH3 55.40 3.76(s) OCH3 55.44 3.86(s) Glucose δC ΔH Rhamnose ΔC δH 1 104.39 4.33(d, 8) 1 102.73 5.18(d, 1) 2 75.66 3.5-4.2 2 72.34 3.5-4.2 3 84.08 3.53(m) 3 72.25 3.5-4.2 4 70.54 3.5-4.2 4 73.99 3.5-4.2 5 75.37 3.5-4.2 5 70.05 3.5-4.2 6 64.48 4.41(m) 6 17.88 1.25(d, 6)

Example 2 Preparation and Quantitative Analysis of Epimeredi indica Root Extract

A: The dried and powdered root of Epimeredi indica was extracted with 10 fold water, and filtered. The residue was extracted with 8 fold water for 2 hours again, and filtered. The filtrates were combined and concentrated under vacuum to obtain the extracts of Epimeredi indica Root.

B: Quantitative Analysis

1. Apparatus and Materials

-   -   Apparatus: Agilent 1100 HPLC system.     -   Standard: Epimeredinoside A     -   Chemical reagents: Methanol, acetonitrile, water and other         chemical reagents were HPLC-grade.     -   Samples: Extracts of Epimeredi indica Root (Shanghai Yaogang         Biotech Co. Ltd)

2. Chromatographic Conditions

-   -   Column: Discovery C18 (250 mm×4.6 mm, 5 μm)     -   Mobile phase: Acetonitrile:Water=27:73     -   Flow rate: 1.0 ml/min     -   Column temperature: Room temperature     -   Detector wavelength: 320 nm     -   Injection volume: 20 μl

3. Calibration Curves

-   -   Preparation of standard stock solutions: The standard was         prepared by weighing 4.95 mg, and dissolving and diluting with         methanol in a 25 ml volumetric flask to obtain standard stock         solutions for the calibration curves.     -   The Calibration Curves: From the stock solutions 0.4, 0.8, 1.2,         1.6, and 2.0 ml were weighed, dissolved, and diluted with         methanol in 2 ml volumetric flask to obtain standard solutions         at concentrations of 39.6 μg/ml, 79.2 μg/ml, 118.8 μg/ml, 158.4         μg/ml, and 198 μg/ml, respectively. A total of 20 μL of each         standard solution was subject to HPLC quantitative analysis. A         calibration curve was generated to confirm the linear         relationship between the peak area ratio (Y axis) and the         concentrations of the standard (X axis) in the test samples. The         calibration curves were found to be linear and could be         described by the regression equations Y=20.139X−154.35, with         coefficient R²=0.9994. The range of calibration curves was         0.792-3.96 μg, and the retention time of epimeredinoside A was         9.55 min.

4. Samples Analysis

Preparation of the standard solutions: The standard was accurately weighed, dissolved, and diluted with methanol in a volumetric flask to obtain standard solutions. A total of 20 μL of standard solution was subject to HPLC quantitative analysis and the peak area was recorded. The contents of epimeredinoside A was calculated using the calibration curves accordingly, see FIG. 2.

Preparation of the sample solutions: The extracts of Epimeredi indica root (176.66 mg) were accurately weighted, and extracted with by ultrasonication at room temperature for 2 times, then centrifuged. The supernatants were combined and diluted with water in a 10 ml volumetric flask. The solution was filtered through a syringe filter (0.45 μm).

The sample solutions were subjected to HPLC analysis as described above, shown in FIG. 3

The content of epimeredinoside A in samples were calculated according to the calibration curves.

Peak area (Y): 383.380.

The concentration X is 26.70 μg/ml according to the regression equations Y=20.139X−154.35.

The content of epimeredinoside A in the sample was 0.15% by the equation X*10/Sample Amount*100%.

Example 3 Preparation of a Granulate

Formula:

Extracts of Epimeredi indica Root 150 g Lactose  50 g Stearate Magnesium  2 g

Methods: The extracts of Epimeredi indica Root which were prepared as described in Example 2 were mixed with lactose and stearate magnesium, and then sieved. The granulate was obtained by sieving again. The content of epimeredinoside A was 0.17%.

Example 4 Preparation of a Granulate

Formula:

Extracts of the Epimeredi indica Root 130 g Lactose  70 g Stearate Magnesium  1 g

Methods: The extracts of Epimeredi indica Root which were prepared as described in Example 2 were mixed with lactose and stearate magnesium, and then sieved. The granulate was obtained by sieving again. The content of epimeredinoside A was 0.13%.

Example 5 Preparation of a Capsule

Formula:

Extracts Epimeredi indica Root 110 g Lactose  90 g Stearate Magnesium  1 g

Methods: The extracts of Epimeredi indica Root which were prepared as described in Example 2 were mixed with lactose and stearate magnesium, and then sieved. The grain was sieved again. The capsules were filled with the fine grain.

The content of epimeredinoside A was 0.27%.

Example 6 Preparation of a Tablet

Formula:

The extracts of Epimeredi indica Root 230 g  Cellulose, Microcrystalline 20 g  Carboxymethyl starch sodium 3 g Polyvinylpyrrolidone 1 g Pulvis Talci 1 g Stearate Magnesium 1 g

Methods: The Microcrystalline Cellulose, Sodium Carboxymethyl Starch and other materials were mixed in a mortar, and the extracts of Epimeredi indica Root which were prepared as described in Example 2 were added. The powder was shaped in a muller. The fine powder was granulated, dried and Magnesium Stearate added. The granulate was tableted and coated. The content of epimeredinoside A was 0.23%.

Example 7 Preparation of a Tablet

Formula:

The extracts of Epimeredi indica Root 300 g  Cellulose, Microcrystalline  26 g Carboxymethyl starch sodium 2.8 g Polyvinylpyrrolidone 2.8 g Pulvis Talci 2.8 g Stearate Magnesium   1 g

Preparation was carried out according to the method mentioned in Example 6. The concentration of epimeredinoside A was 0.22%. 

1. An isolated compound named epimeredinoside A having formula I as follows:


2. An oral pharmaceutical composition comprising an Epimeredi indica root extract containing from 0.10 to 1.50% by weight of epimeredinoside A, wherein the extract has been obtained by extracting Epimeredi indica root with water and concentrating by distillation; and at least one pharmaceutical adjuvant.
 3. The oral pharmaceutical composition according to claim 2, wherein the composition is an oral form selected from the group consisting of hard capsule, soft capsule, granule, tablet, and oral liquid.
 4. A preparation method for preparing oral pharmaceutics from Epimeredi indica root extract, comprising: making a powder of Epimeredi indica roots; adding water to the powder in an amount of about 10 times that of the powder and extracting for a time ranging from 1 to 2 hours; filtering to obtain a first filtrate and a first cake; adding water to the first cake in an amount of about 10 times that of the first cake and extracting for a time ranging from 1 to 2 hours; filtering to obtain a second filtrate and a second cake; combining the first filtrate and the second filtrate to provide a combined filtrate; concentrating the combined filtrate as extracta sicca to a density ranging from 1.01 to 1.08 and a content of epimeredinoside A ranging from 0.10 to 1.50% as determined by HPLC; drying the extracta sicca by spray or vacuum; and mixing predetermined quantities of the dried extract and at least one adjuvant.
 5. The preparation method according claim 4, wherein the content of epimeredinoside A in the dried extract of Epimeredi indica root is determined by HPLC, which comprises the steps of: a. providing (1) an HPLC apparatus, (2) a Standard sample of epimeredinoside A, (3) HPLC grade chemical reagents including methanol, acetonitrile, and distilled water, and (4) extracts of Epimeredi indica root; b. operating the HPLC apparatus under conditions including (1) using a Chromatographic column: Discovery C₁₈ (250 mm×4.6 mm, 5 μm), (2) using a mobile phase which is a mixture of acetonitrile and water having an acetonitrile: water ratio of 27:73, (3) using a flow rate of 1.0 ml/min, (4) using a column temperature which is room temperature, and (5) using a detection wavelength of 320 nm, and (6) using an injection volume of 20 μl; c. generating a calibration curve by (1) preparing standard solutions of epimeredinoside A having respective concentrations of 39.6 μg/ml, 79.2 μg/ml, 118.8 μg/ml, 158.4 μg/ml, and 198 μg/ml; (2) subjecting each standard solution to HPLC quantitative analysis; (3) generating a calibration curve to confirm a linear relationship between peak area ratio (Y axis) and the concentrations of the standard solutions (X axis); d. preparing test samples; and e. subjecting the test samples to the HPLC quantitative analysis; f. determining the content of epimeredinoside A in the test samples from the calibration curves using, as a formula for calculation, Y=20.139X−154.35, where Y is peak area and X is sample concentration (μg/ml). 